EP0060700B1 - Assay method and device - Google Patents

Assay method and device Download PDF

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Publication number
EP0060700B1
EP0060700B1 EP82301285A EP82301285A EP0060700B1 EP 0060700 B1 EP0060700 B1 EP 0060700B1 EP 82301285 A EP82301285 A EP 82301285A EP 82301285 A EP82301285 A EP 82301285A EP 0060700 B1 EP0060700 B1 EP 0060700B1
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EP
European Patent Office
Prior art keywords
analytical
analytical reagent
absorbent
substance
fluid
Prior art date
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Expired
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EP82301285A
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German (de)
English (en)
French (fr)
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EP0060700A1 (en
Inventor
David S. Bauman
Paul A. Barstad
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Immuno Mycologics Inc
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Immuno Mycologics Inc
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Publication date
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Priority to AT82301285T priority Critical patent/ATE16642T1/de
Publication of EP0060700A1 publication Critical patent/EP0060700A1/en
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Publication of EP0060700B1 publication Critical patent/EP0060700B1/en
Expired legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54391—Immunochromatographic test strips based on vertical flow
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/807—Apparatus included in process claim, e.g. physical support structures
    • Y10S436/81—Tube, bottle, or dipstick
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/824—Immunological separation techniques

Definitions

  • This invention relates in general to devices and methods for determination of substances and more particularly to methods and devices for assays of binding or bindable substances. Still further, this invention relates to methods and devices for quantitative assays of such binding or bindable substances.
  • binding and bindable substances of various types.
  • binding and bindable substances and the substances bound thereto or thereby are antibodies- antigens and other binding proteins-protein bindable substances.
  • the insoluble material is separated from the soluble material which includes any unattached enzyme-labelled antigen. Centrifuging or washing the insoluble material achieves this separation. An enzyme- reactive agent is then added to either the insoluble or soluble portions to assay the enzyme activity. In this manner, the presence of unlabelled antigen (the assay substance) is determined.
  • U.S. Patent No. 3,791,932 to Schuurs et al. describes a similar process for assaying either a binding protein or a substance which is specifically bound by such a protein.
  • this assay method one of these components is insolubilized and then mixed with the component to be determined, binding the protein to its complement.
  • the solid phase of the insoluble form is separated and reacted with a coupling product of the substance to be determined with an enzyme; i.e., an enzyme-labelled component is mixed with the solid phase such that the enzyme-labelled component will also be bound.
  • the presence of the component to be determined in the first mixing step will prevent coupling of the coupling product (enzyme-component) with at least some of the insolubilized binding components. Determination of the enzyme activity of either the solid or liquid phase resulting from the last mixture will determine the presence of the described components.
  • U.S. Reissue Patent No. 29,169 is related to U.S. Patent No. 3,791,932.
  • test substance for example, hormone-antibody, antigen-antibody, enzyme-substrate, or their converse reactions, together with direct or indirect quantitative measurement of the amount of reagent so reacted by the measurement of a characteristic of a reagent (or of another substance, an indicator, which has reacted with the reagent) such as color, radioactivity, or other physical characteristics.
  • an appropriate reagent for example, hormone-antibody, antigen-antibody, enzyme-substrate, or their converse reactions, together with direct or indirect quantitative measurement of the amount of reagent so reacted by the measurement of a characteristic of a reagent (or of another substance, an indicator, which has reacted with the reagent) such as color, radioactivity, or other physical characteristics.
  • the reagent is immobilized or fixed to a medium into which test substances can be diffused.
  • test substance is diffused into a gel medium in which reagent is immobilized against diffusion.
  • immobilizing the reagents in the gel is by absorbing the reagent onto talc, porous glass beads, hydroxyapatite, zirconyl phosphate, charcoal polyethylene, polypropylene, polystyrene particles or the like.
  • a conventional assay using a labelled material is obtained by first diffusing test substance into the reagent zone and then using indicators for reacting with the remaining unattached reagent. After excess indicator has been removed, determination of the attached indicator completes the assay.
  • the assay method and device of the present invention are significant improvements over the prior art and it is an object of the present invention to provide improved assay methods and devices, especially with regard to the above- mentioned problems.
  • This invention provides a device for assaying a test substance.
  • This test substance must be capable of being bound by a primary absorbent substance.
  • the primary absorbent substance must be capable of selectively binding an analytical reagent such that the presence or absence of test substance with a predetermined quantity of primary absorbent substance and analytical reagent will prevent some analytical reagent from being bound by the primary absorbent substance.
  • the analytical reagent must be capable of being bound by an analytical absorbent substance.
  • the analytical reagent also must be capable of detection once bound by the analytical absorbent substance. Examples of test substances, primary absorbent substances, analytical reagents, and analytical absorbent substances are set forth below.
  • a device for assaying a test substance comprises a first fluid conducting means having a first opening for receiving fluid therethrough; a predetermined quantity of primary absorbent substance is fixed to a first support means disposed in the first fluid conducting means and to which the primary absorbent substance is attached to retain the primary absorbent substance disposed to contact fluid passing through the first fluid conducting means; second fluid conducting means connected to receive fluid from the first fluid conducting means; and an analytical absorbent substance capable of firmly binding the analytical reagent.
  • the analytical absorbent substance is supported by a second support means disposed in the second fluid conducting means.
  • the second support means retains the analytical absorbent substance within the second fluid conducting means.
  • Means for detecting the analytical reagent absorbed by the analytical absorbent substance is provided.
  • the second support means supports the analytical absorbent substance in a series of continuous or discontinuous zones through which the fluid sequentially passes such that analytical reagent is sequentially bound therein as it passes therethrough.
  • a predetermined quantity of analytical absorbent is disposed in each zone for binding only a predetermined quantity of analytical reagent.
  • detecting the last zone in which analytical reagent is bound indicates the quantity of analytical reagent absorbed in the secondary support means.
  • an assay for a test substance comprises steps utilizing the substances described above. First a predetermined quantity of analytical reagent and a test substance is conducted through and contacted with a predetermined quantity of primary absorbent substance such that the presence or absence of test substance will cause a quantity of analytical reagent not to be bound by the primary absorbent substance. Second, any analytical reagent not bound by the primary absorbent substance is received by and contacted with an analytical absorbent substance binding the analytical reagent to the analytical absorbent substance. The presence of analytical reagent bound to the analytical absorbent substance is then determined.
  • the analytical reagent is sequentially conducted through a series of zones containing predetermined quantities of analytical absorbent and the quantity of bound analytical reagent is determined by detecting the last zone in which analytical reagent is bound.
  • this method can provide a quantitative assay for test substance.
  • the present invention utilizes a primary absorbent substance, an analytical reagent substance, and an analytical absorbent substance.
  • a primary absorbent substance an analytical reagent substance
  • an analytical absorbent substance an analytical absorbent substance.
  • Each of these substances are specifically related to each other and to the test substance. Furthermore, each must have certain properties in order for the assay and the assay device to function.
  • test substance must be capable of being specifically bound by the primary absorbent. It must also be capable of being bound by the primary absorbent in such a way that, when mixed with a specific amount of primary absorbent and a specific amount of analytical reagent, some of the analytical reagent will or will not be bound.
  • the amount of analytical reagent bound or not bound should be proportional to the amount of test substance mixed with the primary absorbent and analytical reagent.
  • the analytical reagent must be capable of being specifically bound by a primary absorbent or by a primary absorbent to which test substance is bound. It must also be capable of being specifically bound by an analytical absorbent.
  • the analytical reagent must be capable of being bound by the primary absorbent in such a way that, when a specific amount of analytical reagent is mixed with a specific amount of primary absorbent and at least some test substance, some of the analytical reagent will or will not be bound by the primary absorbent.
  • the analytical reagent must also be capable of detection once bound by the analytical absorbent.
  • the analytical reagent is bound by the analytical absorbent in a very fast reaction so that a distinct and accurate sequential zone measurement can be detected after binding. The sequential zone measurement of analytical absorbent is described in more detail below.
  • the primary absorbent must be capable of specifically binding test substance as described above. It must also be capable of specifically binding analytical reagent or capable of binding analytical reagent when test substance is attached thereto.
  • the amount of analytical reagent bound or not bound should be proportional to the amount of test substance mixed with the primary absorbent.
  • the primary absorbent should be fixed in a specific location such that analytical reagent and test substance can pass through and be contacted with the primary absorbent.
  • the analytical absorbent must be capable of specifically binding analytical reagent. And, in the assay device the analytical reagent should be fixed in a specific location. Preferably predetermined quantities of the analytical reagent should be fixed in a series of zones through which fluid sequentially passes such that analytical reagent will be sequentially bound in the zones.
  • Test substances which meet the requirements for a test substance include antigens, antibodies, and various other organic substances. Since the most important requirement of the test substance is that the test substance be capable of being specifically bound by a primary absorbent, those skilled in the art will recognize that a primary absorbent for specifically binding most substances can be prepared.
  • primary absorbent substances having the properties described above, are possible.
  • the primary absorbent will depend upon the test substance which must be bound by the primary absorbent.
  • primary absorbents for antigens or organic molecules assays would include antibodies and lectons specific for the molecule to be assayed.
  • antibodies against specific microbial antigens, proteins, hormones, polysaccharides, antibodies, toxins, etc. could be used for a primary absorbent substance.
  • lectins with specific binding activity for sugars, enzyme cofactors, polysaccharides, etc. could be used.
  • Primary absorbent substances for antibody assays could include antigens or organic compounds for which the antibody is specific.
  • microbial antigen for example, microbial antigen, protein, hormomes, polysaccharides, antibiotic, toxin, etc. having specific binding activity with the antibody to be assayed could be used; binding protein and bindable substance, e.g. protein are also suitable.
  • a wide variety of chemicals can also be used for analytical reagents.
  • One simple way of preparing an analytical reagent is to utilize the substance which is being tested for by labelling the test substance chemical with a detectable group and with a group capable of binding specifically to the analytical absorbent.
  • the test substance is an antibody
  • the antibody can be coupled to an enzyme for detection and also coupled to biotin for specifically binding to an avidin analytical absorbent.
  • detectable groups can be coupled to particular compounds in forming the analytical reagent.
  • detectable or indicator groups include colored dye, enzymes, fluorescent compounds, radioisotopes, and others.
  • Particular enzymes include horse radish peroxidase, alkaline phosphate, B-galactosidase and others.
  • colored dyes are amido black, and eosin.
  • Fluorescent compounds include fluorescein isothiocyanate, dansyl, and others.
  • Radioisotopes include Carbon 14 and Iodine 125 and others.
  • the detectable group most desirable depends upon the particular assay and the particular device being utilized for the assay; enzymes are preferred.
  • the group coupled to the analytical reagent capable of specifically binding to the analytical absorbent depends upon the analytical absorbent.
  • binding groups capable of binding specifically to the analytical absorbent are biotin; avidin; antigen; enzyme substrate analogues; antibody; 2,4-dinetrophenol (specific for MOPC-315 mouse myeloma protein); and enzyme; avidin and biotin are preferred.
  • Compounds to which the compounds described above are bound can include antibodies, antigens, organic molecules and lectins.
  • the antibodies may be conventional antibodies or they may be monoclonal antibodies specific against microbial antigens, proteins, polysaccharides, antibiotics, toxins, hormones, etc.
  • the antigens may be microbial antigens, proteins, polysaccharides, antibiotics, toxins, hormones, etc.
  • the organic molecules include all of those capable of specific binding to lectin.
  • the analytical reagent While a preferred method of forming the analytical reagent is to couple groups to a compound with the same specificity as the material being assayed with respect to the primary absorbent, this is not an absolute requirement. As described in more detail below, the analytical reagent may have specific binding activity with the test substance and not the primary absorbent alone. In this instance, the analytical reagent will pass through the primary absorbent if no test substance is coupled thereto.
  • a particularly useful analytical absorbent consists of either avidin or biotin.
  • Avidin couples to biotin with a very fast coupling rate.
  • avidin and biotin are widely available and relatively inexpensive.
  • avidin and biotin can be readily coupled to various types of supports and to many other components.
  • the apparatus 11 consists of a clear glass test tube 13 having an opening at its upper end.
  • the top of tube 13 is press-fitted into a lip which extends downwardly from the upper rim of a funnel 17.
  • the funnel 17 extends downwardly into the tube 13 and has a cylindrical receptacle 19 at its lower end.
  • the cylindrical receptacle 19 receives, press-fitted therein, a clear glass capillary tube 21.
  • the capillary tube 21 is filled with a plurality of glass beads 23. These glass beads 23 fixedly support the analytical absorbent within the capillary tube 21 for contacting fluid passing through the capillary tube 21.
  • a plug 25 is press-fitted into the lower end of capillary tube 21 to retain the beads 23 within the tube 21.
  • a plug 27 is press-fitted into the upper end of the capillary tube 21 to retain the beads 23 within tube 21 when inverted.
  • the capillary tube 21 is held suspended inside test tube 23 by funnel 17. Fluid passing through capillary tube 21 will pass into and be retained by test tube 13. The beads 23 and the interior of capillary tube 21 are visible through the walls of test tube 13 and capillary tube 21.
  • a lower cylindrical lip 15 and an upper cylindrical lip 29 Extending from the upper rim of funnel 17 is a lower cylindrical lip 15 and an upper cylindrical lip 29. These lips extend axially.
  • the lower lip 15 receives the upper end of glass tube 13 and extends outside test tube 13 with tube 13 press-fitted therein. If desired, glue or the like can permanently bond tube 13 to funnel 17.
  • Upper lip 29 receives a cup 31. Cup 31 has the same cylindrical diameter as tube 13. The cup 31 is press-fitted into the upper cylindrical lip 29 in the same manner as tube 13 is press-fitted into the lower cylindrical lip 15.
  • Cup 31 is disposed above a shoulder rim 33 of funnel 17 extending inwardly between lip 25 and lip 27. Cup 31 has openings in its upper end and its lower end.
  • the interior of funnel 17 between plugs 27 and 35 is filled with a plurality of glass beads 37.
  • the beads 37 fixedly support the primary absorbent for contacting fluid passing through funnel 17.
  • the plugs 25, 27 and 35 are each made of a porous material such as polypropylene which fluid can readily pass but firmly retaining the beads within funnel 17 and tube 21.
  • Plug 27 separates the beads 37 and the beads 23.
  • cup 31 and plug 35 can be fused or glued to funnel 17 to premanently hold them in position.
  • a threaded cap 39 is received by threads 41 at the upper end of cup 31.
  • Cap 39 closes the upper end of cup 31 and prevents foreign matter from inadvertently entering the assay device.
  • the construction of the device 11 provides a specific fluid conducting passageway such that fluid enters through the top of cup 31, passes through plug 35 and contacts beads 37, through plugs 27 into capillary 21 contacting beads 23, and then passes through the plug 25 and into tube 13.
  • fluid it is important that the fluid be first intimately contacted with and passed through beads 37 prior to being conducted through capillary 21. It is also important that capillary 21 be a relatively narrow path. This allows sequential binding to the beads 23 and the analytical absorbent. By arrangement of the beads in zones and by loading these zones with predetermined amounts of analytical absorbent, sequential binding of analytical reagent can be quantitatively determined by the last zone in which binding occurs.
  • graduation marks 43 can be placed on the capillary tube 21 to allow visual examination and determination of the length of linear reaction (or the last zone in which binding of analytical reagent has occurred) within the tube. Also if desired, a small hole can be provided through funnel 17 or tube 13 to allow air to escape as liquid enters tube 13 through capillary 21.
  • beads 23 and 37 support the primary absorbent substance and analytical absorbent, respectively. Beads are utilized because they insure that fluids passing therethrough will have an intimate contact with the absorbent substance disposed thereon. It is also relatively easy to attach absorbent substance to beads so that the respective absorbent substance will not escape funnel 17 and capillary tube 21.
  • Figs. 1 and 2 show the primary absorbent substance and analytical absorbent being supported on glass beads
  • supports can include membrane filters such as cellulose acetate, or cellulose acetate-nitrate; glass fiber filters; or beads made of agarose, cellulose, dextran, polyacrylamide, etc.
  • membrane filters such as cellulose acetate, or cellulose acetate-nitrate
  • glass fiber filters such as cellulose acetate-nitrate
  • no separate support would be required if a sufficiently narrow tube is utilized and the absorbent is attached to the inner wall of the tube.
  • the main feature of the support material is that it provides intimate contact between the absorbent substance attached thereto and the fluid passing through the fluid passageway.
  • the capillary tube 21 is slightly oversized to make clear its detail. Normally, the interior diameter of the capillary tube is approximately .5 to 2 mm.
  • the glass beads 23 and ' 37 each have a diameter of approximately .01 to 5 mm. The diameter of the capillary tube 21 and the diameter of the glass beads each depends upon the density of the absorbent, the desired sensitivity of the quantitative measurement, and the desired flow rate for fluid through the capillary 21.
  • proteins and polysaccharides can be coupled onto supports such as cellulose filters, cellulose acetate/nitrate filters, nylon filters, agarose beads, cellulose beads, and polyacrylamide beads. It can also be used for the introduction of spacer groups such as diamino alkyl or aryl, or gamma amino alkyl or aryl carboxylic acid onto the support. Briefly, the sequence of reaction is as follows:
  • various triethoxysilanes and trimethoxysilanes may be used.
  • gamma-aminopropyltriethoxysilane or N - (B - aminoethyl - gamma - aminopropyl)trimethoxysilane as spacer groups attached to glass as follows:
  • Carbodiimide coupling of other proteins and other amine containing organic molecules to spacer groups on supports may be achieved as follows:
  • Carbodiimide coupling of organic molecules containing carboxyl or aldehyde groups to spacer groups on supports may be achieved as follows:
  • Gluteraldehyde coupling or proteins and other amine-containing molecules to spacer groups on supports may be achieved as follows:
  • an apparatus as described above can be utilized.
  • a predetermined quantity of primary absorbent is attached to the beads 37.
  • a predetermined density of analytical absorbent is prepared through the column of glass beads 23 in capillary tube 21.
  • the primary absorbent and the analytical absorbent are prepared specifically for a particular test substance and analytical reagent.
  • a predetermined amount of analytical reagent is prepared for use with the apparatus.
  • the analytical reagent is of a type specifically bound by a primary absorbent so that analytical reagent competes with test substance for being bound by the primary absorbent.
  • a specific quantity of fluid to be assayed for test substance is mixed with a predetermined quantity of analytical reagent. This mixture is then poured into cup 31. The test substance and the analytical reagent pass through and contact the primary absorbent substance held on beads 37. If test substance is present in the assay fluid, some of the analytical reagent will not be bound by the primary absorbent and will pass through the beads 37 and into the capillary tube 21. The amount of analytical reagent which passes through the beads 37 into the tube 21 is proportional to the amount of test substance bound by the primary absorbent.
  • the specific amounts of primary absorbent on beads 37 and the amount of analytical reagent mixed with the assay fluid is carefully balanced to insure that no analytical reagent will pass through the beads 37 if no test substance is present, and, at the same time, at least some analytical reagent will pass through the beads 37 if test substance is present in the assay fluid.
  • analytical reagent passes through the beads 37 and the primary absorbent it will enter capillary tube 21 and be bound by the analytical absorbent substance disposed on the beads 23 therein. If the binding reaction between analytical reagent and analytical absorbent is sufficiently fast, the binding of analytical reagent will occur linearly down the column of analytica! absorbent. For example, if only enough analytical reagent is present to bind one third of the analytical absorbent in the capillary tube 21, all of the binding will occur in the upper one-third of the capillary tube 21. None of the bottom two-thirds of the capillary tube will have any analytical reagent bound therein.
  • the quantity of analytical reagent bound, and hence, the quantity of test substance in the assay fluid can be determined by a visual inspection of the capillary tube 21.
  • the length of the graduated capillary tube colored by the dye must be compared to the density of the analytical absorbent and the proportionality of analytical reagent which is not bound due to the presence of test substance. This relationship can be empirically or analytically determined.
  • the analytical reagent contains an enzyme
  • an enzyme reactive agent can be passed through the analytical absorbent tube to assay the enzyme activity.
  • the product of the enzyme reactive agent and the enzyme will be fixed in the column due to the physical size of the openings between the glass beads 23.
  • a fluorescent compound is utilized in the analytical reagent
  • the column of analytical absorbent can be surveyed for fluorescence after the analytical reagent is bound.
  • radioisotopes are utilized in the analytical reagent, the column of analytical absorbent can be surveyed for the presence of radioisotopes after the analytical reagent has been found.
  • the fluids need not necessarily be mixed nor is the particular order in which they contact the primary absorbent required.
  • the analytical reagent could be poured through the beads 37 first followed by assay fluid.
  • the assay could also be performed in reverse order; assay fluid first, analytical reagent second.
  • the order is not critical in most cases because, generally, test substance will displace analytical reagent bound by the primary absorbent if the analytical reagent is bound first. In contrast, generally, test substance will not be displaced by analytical reagent if the test substance is bound first.
  • the order of adding analytical reagent and assay fluid can be important. For example, if the analytical reagent is not specifically bound by the primary absorbent alone, but is only bound by primary absorbent to which test substance has been bound, then the assay fluid must be added first followed by the analytical reagent. Furthermore, in this type of assay, it is the amount of analytical reagent bound by the beads 37 and not bound in the beads 23 which is proportional to the amount of test substance.
  • An example of such an assay is an assay for a specific antibody where the primary absorbent is an antigen specific for this antibody and the analytical reagent is an anti antibody specific for the particular antibody but not for the antigen.
  • the assay fluid (containing antibody) must be contacted with the primary absorbent (antigen) and then the analytical reagent (anti antibody - detectable substance - substance bound by analytical absorbent) contacted with the primary absorbent. Or at least, the analytical reagent cannot be contacted with the primary absorbent, alone, first.
  • This concept of binding analytical reagent directly to bound test substance can be utilized to provide a slightly different type of assay.
  • the primary absorbent can be of specificity only to the test substance. This would allow even a non-chemical primary absorbent such as filters or the like if the test substance can be bound thereby while allowing the analytical reagent to pass therethrough if no test substance is bound.
  • the method and device of the present invention provide a quantitative assay much improved over assays which require color determination or intensity determinations.
  • the present invention can be made as sensitive as desired by regulating the linear density of the analytical absorbent as well as the sensitivity of the detection means for the analytical reagent. Extremely sensitive quantitative assays can be performed with enzyme-labelled analytical reagents and fine linear densities of analytical absorbent.
  • Another way of providing a sensitive test while utilizing a straight, linear density is to run successive tests with successively smaller quantities of assay fluid being mixed with the analytical reagent. For example, if the first run of an assay produces a column wherein analytical absorbent is completely bound with analytical reagent a second test can be run utilizing one-tenth the quantity of assay fluid. Successively decreased amounts of assay fluid can be added until only a portion of the analytical absorbent is bound with analytical reagent. The quantitative density of test substance in the assay fluid can be scaled accordingly.
  • the present invention and method for assaying do not require washing to separate an insolubilized component from a labelled component. This is because the primary absorbent is fixed on a permeable support. The analytical reagent not bound by the primary absorbent passes through the support material simply by gravity. Of course, suction could be applied to provide a faster flow of the fluid.
  • the present invention can be used with special advantage with the recently produced hybridoma or monoclonal antibodies. Production of such antibodies is described in U.S. Patent No. 4,196,265 to Koprowski et al.
  • a particular advantage of monoclonal antibodies is that they are readily produced against specific virus and antigenic determinants.
  • assay methods and devices can be provided which are precisely specific for a particular determinant and are quantitatively accurate as well.
  • the device is both portable and has a long shelf life. This allows field assays to be accurate in quantity and allows the assay devices to be stored for long periods of time.
  • the portability and long shelf life are provided by the stable chemical binding of the analytical absorbent and primary absorbent on the membranes and beads used as supports. Generally, these chemicals can be allowed to dry without adverse effect.
  • the analytical reagent can be stored separately and will also have a long shelf life.
  • zone length tended to have a linear proportionality to analytical reagent and, hence, test substance.
  • the first fluid conducting means comprises a funnel for catching and directing a downwardly flowing fluid to a lower opening in said funnel to which the second fluid conducting means is attached;
  • the second fluid conducting means preferably comprises a narrow, transparent tube.
  • the device preferably further comprises transparent housing means for housing the funnel and the transparent tube and for receiving fluid which drains from the transparent tube.
  • the support means preferably comprise beads.
  • the support means are beads and the device further comprises:
  • test device and method of the invention are particularly suitable when the test substance is an organic substance capable of being bound by an antibody, antigen, binding protein, bindable protein, polysaccharide, antibiotic, hormone or toxin.
  • the analytical reagent used in the device and method of the invention is preferably a compound incorporating:

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EP82301285A 1981-03-13 1982-03-12 Assay method and device Expired EP0060700B1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AT82301285T ATE16642T1 (de) 1981-03-13 1982-03-12 Untersuchungsverfahren und -vorrichtung.

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US06/243,547 US4425438A (en) 1981-03-13 1981-03-13 Assay method and device
US243547 1981-03-13

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EP0060700A1 EP0060700A1 (en) 1982-09-22
EP0060700B1 true EP0060700B1 (en) 1985-11-21

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US (1) US4425438A (enrdf_load_stackoverflow)
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JPH0459586B2 (enrdf_load_stackoverflow) 1992-09-22
US4425438A (en) 1984-01-10
ATE16642T1 (de) 1985-12-15
JPS57161647A (en) 1982-10-05
AU560649B2 (en) 1987-04-16
KR830009486A (ko) 1983-12-21
DE3267501D1 (en) 1986-01-02
CA1184114A (en) 1985-03-19
EP0060700A1 (en) 1982-09-22
KR880000359B1 (ko) 1988-03-20
AU8135482A (en) 1982-09-16

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